Yuqing Zhao

Isolation, Structural Determination, and Evaluation of the Biological Activity of 20(S)-25-methoxyl-dammarane-3β, 12β, 20-triol [20(S)-25-OCH3-PPD], a Novel Natural Product from Panax notoginseng

Ginseng has been used extensively for medicinal purposes, with suggested utility for indications as diverse as diabetes, cardiovascular disease and cancer. Herein we report the discovery and characterization of 20(S)-25-OCH3-PPD, a ginsenoside that inhibits growth and survival of cancer cells. The novel dammarane triterpene sapogenin (C31H56O4; molecular weight 492) was isolated from the total hydrolyzed saponins extracted from the leaves of Panax notoginseng using conventional and reverse-phase silica gel chromatography. Based on physicochemical characteristics and NMR data, the compound was identified as 20(S)-25-OCH3-PPD. The biological activities of 20(S)-25-OCH3-PPD and its known analogs, 20(S)-PPD and Rg3, were evaluated in 12 human cancer cell lines. In all cell lines, the order of cytotoxicity of the test compounds was 20(S)-25-OCH3-PPD >> 20(S)-PPD >> Rg3. 20(S)-25-OCH3-PPD also induced apoptosis and cell cycle arrest in the G1 phase, and inhibited proliferation in breast cancer cell lines, demonstrating its potent biological effects. In regard to cytotoxicity, the IC50 values of 20(S)-25-OCH3-PPD for most cell lines were in the lower microM range, a 5-15-fold greater cytotoxicity relative to 20(S)-PPD and a 10-100-fold increase over Rg3. These findings suggest a structure-activity relationship among dammarane-type sapogenins. The data presented here may provide a basis for the future development of 20(S)-25-OCH3-PPD as a novel anti-cancer agent.

Polyketide butenolide, diphenyl ether, and benzophenone derivatives from the fungus Aspergillus flavipes PJ03-11

Five new polyketides including three new butenolides (1-3), one new diphenyl ether (4), and one new benzophenone (5), together with eleven known compounds (6-16) were isolated from a wetland fungus Aspergillus flavipes PJ03-11. Their structures were elucidated on the basis of detailed spectroscopic analysis. All the isolated compounds were tested for their α-glucosidase inhibitory activities. The results showed that compounds 1-3, 6, 7, 11, 15 and 16 exhibited stronger inhibitory activities than acarbose. And the preliminary structure-activity relationships of aspulvinone and diphenyl ether compounds on the α-glucosidase inhibitory activity were reported.

Flavonoids and its derivatives from Callistephus chinensis flowers and their inhibitory activities against alpha-glucosidase

Inhibitors of carbohydrate-hydrolysing enzymes play an important role for the treatment of diabetes. One of the therapeutic methods for decreasing of postprandial hyperglycemia is to retard absorption of glucose by the inhibition of carbohydrate- hydrolysing enzymes, such as α-glucosidase, in the digestive organs. To investigate the therapeutic potential of compounds from natural sources, Callistephus chinensis flowers (CCF) were tested for inhibition of α-glucosidase, and acarboes was used as the positive control. The 70 % ethanol extract of CCF exhibited significant α-glucosidase inhibitory activities with IC50 value of 8.14 μg/ml. The stepwise polarity fractions of CCF were tested further for in vitro inhibition of α-glucosidase. The ethyl acetate (EtOAc) fraction exhibited the most significant inhibitory activity. Eight pure compounds, apigenin, apigenin-7-O-β-D- glucoside, kaempferol, hyperin, naringenin, quercetin, luteolin, and kaempferol-7-O-β-D- glucoside, were isolated (using enzyme assay-guide fractionation method) from the EtOAc fraction. Among these, quercetin was the most active one (IC50 values 2.04 μg/ml), and it appears that the inhibiting percentages are close to acarbose (IC50 values 2.24 μg/ml), the positive control, on α-glucosidase inhibition. HPLC/UV analysis indicated that the major components of CCF are kaempferol, hyperin and quercetin. The presented results revealed that CCF containing these eight flavonoids could be a useful natural source in the development of a novel α-glucosidase inhibitory agent against diabetic complications.

Antitumor activity of ginseng sapogenins, 25-OH-PPD and 25-OCH3-PPD, on gastric cancer cells

Objectives25-Hydroxyprotopanaxadiol (25-OH-PPD) and 25-methoxylprotopanaxadiol (25-OCH3-PPD), two ginseng sapogenins, have potent antitumor activity and their effects on gastric cancer (BGC-823, SGC-7901, MKN-28) cells and a gastric mucosa (GES-1) cell line are reported.ResultsBoth compounds significantly inhibited the growth of gastric cancer cells, while having lesser inhibitory effects on GES-1 cells by MTT assay. A mechanistic study revealed that the two ginseng sapogenins could induce apoptosis in BGC-823 cells by morphological observation, DNA fragmentation, flow cytometry and western blot analysis. Besides, the apoptosis was inhibited by Ac-DEVD-CHO, a caspase 3 inhibitor, which was confirmed by cell viability analysis.ConclusionsThese results indicate that 25-OH-PPD and 25-OCH3-PPD have potential to be promising agents for the treatment of gastric cancer.

In Vitro Metabolism of 20(R)-25-Methoxyl-Dammarane-3, 12, 20-Triol from Panax notoginseng in Human, Monkey, Dog, Rat, and Mouse Liver Microsomes

The present study characterized in vitro metabolites of 20(R)-25-methoxyl-dammarane-3β, 12β, 20-triol (20(R)-25-OCH3-PPD) in mouse, rat, dog, monkey and human liver microsomes. 20(R)-25-OCH3-PPD was incubated with liver microsomes in the presence of NADPH. The reaction mixtures and the metabolites were identified on the basis of their mass profiles using LC-Q/TOF and were quantified using triple quadrupole instrument by multiple reaction monitoring. A total of 7 metabolites (M1–M7) of the phase I metabolites were detected in all species. 25(R)-OCH3-PPD was metabolized by hydroxylation, dehydrogenation, and O-demethylation. Enzyme kinetic of 20(R)-25-OCH3-PPD metabolism was evaluated in rat and human hepatic microsomes. Incubations studies with selective chemical inhibitors demonstrated that the metabolism of 20(R)-25-OCH3-PPD was primarily mediated by CYP3A4. We conclude that 20(R)-25-OCH3-PPD was metabolized extensively in mammalian species of mouse, rat, dog, monkey, and human. CYP3A4-catalyzed oxygenation metabolism played an important role in the disposition of 25(R)-OCH3-PPD, especially at the C-20 hydroxyl group.

Improved oral bioavailability of 20(R)-25-methoxyl-dammarane-3β, 12β, 20-triol using nanoemulsion based on phospholipid complex: design, characterization, and in vivo pharmacokinetics in rats

The aim of the study was to improve the oral absorption of the compound 25-OCH3-PPD with poor hydrophilicity and lipophilicity. 25-OCH3-PPD-phospholipid complex was prepared by solvent evaporation, then characterized by differential scanning calorimetry, scanning electron microscopy, and infrared absorption spectroscopy. The aqueous solubility and oil–water partition coefficient were compared with the free compound. A nanoemulsion loaded with 25-OCH3-PPD-phospholipid complex was developed by dissolving the complex in water in the presence of hydrophilic surfactant under sonication. After oral administration of the nanoemulsion and the suspension of 25-OCH3-PPD in rats, the concentrations of 25-OCH3-PPD in plasma were determined by high-performance liquid chromatography–tandem mass spectrometry method. The results showed that the solubility of the complex in water and n-octanol was enhanced. The oil–water partition coefficient improved 1.7 times. Peak plasma concentration and area under the curve(0–24 h) of the nanoemulsion of 25-OCH3-PPD-phospholipid complex were higher than that of free compound by 3.9- and 3.5-folds.

Determination of 25-OCH3-PPD and the related substances by UPLC–MS/MS and their cytotoxic activity

20(R)-25-methoxyl-dammarane-3β,12β,20-triol (25-OCH3-PPD) is a promising antitumor compound belonging to triterpenoid saponins isolated from radix notoginseng. A systematic research on the related impurities in raw material of 25-OCH3-PPD has not been conducted. In this study, three impurities obtained by HPLC-ELSD and characterized by (13)C NMR and MS were observed in the raw material of 25-OCH3-PPD. Cytotoxic activities of the related substances were also evaluated, of which impurity B with 25-OCH3-PPD showed synergistic inhibitory activity against BGC-823 with IC50 values of 8.33μM. Furthermore, a rapid and selective UPLC-MS/MS method was developed for simultaneous determination of the principal component and three related substances in the raw material of 25-OCH3-PPD. Multiple reaction monitoring scan mode was used for the quantification of 20(R)-25-OCH3-PPD and its three related substances. The four constituents were separated within 11min on a BEH C18 column (100 mm×2.1mm, 1.7μm) using a mobile phase comprising methanol and 0.03% formic acid water (82:18, v/v) at a flow rate of 0.2mL/min. The proposed UPLC-MS/MS method displayed acceptable levels of linearity, precision, repeatability, and accuracy. In addition, the proposed method was successfully applied for the establishment of a rational quality control standard for the raw material of 25-OCH3-PPD.

Design, characterization, and in vitro antiproliferative efficacy of gemcitabine conjugates based on carboxymethyl glucan

Gemcitabine (GEM) is widely used in clinical practice in the treatment of cancer and several other solid tumors. Nevertheless, the antitumor effect of GEM is partially prevented by some limitations including short half life, and lack of tumor localizing. Carboxymethyl glucan (CMG), a carboxymethylated derivative of β-(1-3)-glucan, shows biocompatibility and biodegradability as well as a potential anticarcinogenic effect. To enhance the antiproliferative activity of GEM, four water soluble conjugates of GEM bound to CMG via diverse amino acid linkers were designed and synthesized. 1H NMR, FT IR, elementary analysis and RP-HPLC chromatography were employed to verify the correct achievement of the conjugates. In vitro release study indicated that conjugates presented slower release in physiological buffer (pH 7.4) than acidic buffer (pH 5.5) mimicking the acidic tumor microenvironment. Moreover, A549, HeLa and Caco-2 cancer cell lines were used to evaluate the in vitro cytotoxicity of conjugates and the results showed that binding GEM to CMG significantly enhanced antiproliferative activity of GEM on A549 cells. Therefore, these conjugates may be potentially useful as a delivery vehicle in cancer therapy and worthy of further study on structure-activity relationship and antiproliferative activity in vitro and in vivo, especially for lung tumor.

Synthesis and anti-tumor evaluation of panaxadiol halogen-derivatives

In the current work, 13 novel panaxadiol (PD) derivatives were synthesized by reacting with chloroacetyl chloride and bromoacetyl bromide. Their in vitro antitumor activities were evaluated on three human tumor cell lines (HCT-116, BGC-823, SW-480) and three normal cells (human gastric epithelial cell line-GES-1, hair follicle dermal papilla cell line-HHDPC and rat myocardial cell line-H9C2) by MTT assay. Compared with PD, the results demonstrated that compound 1e, 2d, 2e showed significant anti-tumor activity against three tumor cell lines, the IC50 value of compound 2d against HCT-116 was the lowest (3.836μM). The anti-tumor activity of open-ring compounds are significantly better than the compounds of C-25 cyclization. Compound 1f, 2f, 2g showed the strong anti-tumor activity. The IC50 value of compound 2g against BGC-823 and SW-480 were the lowest (0.6μM and 0.1μM, respectively). Combined with cytotoxicity test, the IC50 value of compound 1e, 2d, 2e are greater than 100. the open-ring compounds (1f, 2f, 2g) showed a strong toxicity. The toxicity of 1f is lower than 2f and 2g. These compounds may be useful for the development of novel antiproliferative agents.

Synthesis of novel 25-hydroxyprotopanaxadiol derivatives by methylation and methoxycarbonylation using dimethyl carbonate as a environment-friendly reagent and their anti-tumor evaluation

A previous study involving 25-hydroxyprotopanaxadiol (25-OH-PPD) illustrated that the anti-cancer activity increased by 1-3 times after C-3/C-12-OH was substituted by short-chain fatty acids. In addition, 25-OCH3-PPD was also one of our research interests; the unique difference in structure between 25-OH-PPD and 25-OCH3-PPD is that in C-25, the latter activity was 2-5 times higher than that of 25-OH-PPD. These data serves as the scientific basis of our continuing research. To further confirm the effect of short chain acylated and methylated products on the activity and to identify more potent, higher selectivity compounds, we modified 25-OH-PPD with a green environment-friendly and non-toxic chemical dimethyl carbonate (DMC), which plays the role of both solvent and reagent. This experiment yielded 14 derivatives. Their in vitro anti-tumor activities were tested on two different human tumor cell lines (HeLa and DU145) and one normal cell line (IOSE144) by standard MTT assay. The results showed that compounds 3, 5, 6, 10, 11, 12, and 13 exhibited higher cytotoxic activity on two cell lines, with IC50 values within the range of 1.1-12μM. Compounds 12 and 13 exhibited the highest potent activity, with IC50 values of 1.1 and 1.2μM, respectively, on HeLa cells. Antitumor activity significantly increased after the hydroxyl groups are substituted by methyl. The results of the present study may provide useful data for evaluating the structure-activity relationships of other dammarane-type sapogenins and developing new antitumor agents.